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能不能成为好蛋白，生活环境很重要

发布: 2016-03-28 715阅读 205评论

和蛋白打了好些年的交到，也多少了解了些他们的脾气，他们有些时候确实敏感细腻，稍有招待不周，就集体罢工、组团报废。不论是纯化的或是混合的，总之让蛋白们高兴地活着是一件很重要的事，通常情况下，这就是说要让这些蛋白不聚结、能溶解而且有活性。那么我们就需要给蛋白营造一个好的生活环境，也就是一个适当的缓冲液（buffer）同模拟生物细胞内部的稳态。在选择一个适当的缓冲液前，有几点是需要考虑的：1）缓冲液的pH；2）缓冲液的种类；3）盐浓度；4）还原剂；5）稳定剂。

pH

一般情况下，蛋白是要待在pH7.4的环境下，目的是模拟细胞稳态以了解蛋白在细胞内的功能。如果你的蛋白在这个pH下是稳定的，那么pH7.4就是个不错的选择。如果他们不稳定，则需要调整缓冲液的pH。一般经验告诉我们，当pH=pI的时候，即pH就是这个蛋白等电位点的值得时候，蛋白不太容易溶解（不知道什么是pI的，自行百度吧>^<）。查pI值的快速方法就是使用ExPASy ProtParam 进行计算（http://web.expasy.org/protparam/）。

缓冲液种类

一旦决定了pH值，接下来第一件事就是选择一个你要用的缓冲液，一定要保证这个缓冲液在所选pH上能够有缓冲能力，所以所选缓冲液的pKa（酸度系数）最好能在所选pH值所代表的酸电离度的附近。第二件事就是确定缓冲液的浓度，一般浓度是50-100 mM，有时候缓冲液浓度不能太高，以免影响蛋白的活性。第三件事就是注意到一些蛋白和缓冲液的特性，比如，磷酸基团会影响到激酶的活性，所以在溶解激酶的时候，要避免使用带磷酸基团的缓冲液，或者要把磷酸基团透析出去；再比如，很多缓冲液的pH是和温度有很大关系的，有时候在常温下配的缓冲液达到了我们想要的pH，但我们把缓冲液保存在4度时或是蛋白反应要在37度，pH变化会很大，最有名的就是Tri 缓冲液，pH会很大程度上受到温度影响。

盐浓度

常用于各种蛋白缓冲液的盐自然是NaCl，使用盐的目的是让蛋白质更容易溶解在缓冲液里，同时模拟蛋白生活的生理环境（physiological conditions of cells）。小编一般用150 mM NaCl在蛋白缓冲液中，但是根据情况的不同，盐浓度是可能会改变的。比如说不同阶段的蛋白纯化步骤，就像用离子交换柱纯化特定蛋白的时候，我们总归是先用低盐的缓冲液把蛋白输送进虑柱，而使用高盐缓冲液滤出蛋白。升高盐浓度是十分容易的事情，即配比缓冲液时多溶些盐；降低盐浓度，则通常使用透析的方法。

还原剂

如果目标蛋白含有半胱氨酸（cysteine）这样的氨基酸，那么防止-SH基团被氧化而导致的蛋白聚合是十分必要的。那么我们的缓冲液中要按情况添加一定浓度的还原剂。常见的还原剂有DTT（Dithiothreitol）、TCEP（tris(2-carboxyethyl)phosphine）或者2-mercaptpethanol（BME）。TCEP（tris(2-carboxyethyl)phosphine）是这三种中最稳定也是最贵的。实验室里常见的是DTT和BME这两种，小编一般会把这些还原剂的浓度保持在5-10 mM左右（基本上是要比蛋白浓度要高的）。由于DTT和BME，小编一般是配置无还原剂的缓冲液，等到要用的当天，把储存在冰箱里的DTT或BME按一定比例配比到缓冲液里。特别要注意的是，在某些蛋白纯化的时候，一定要查看所用虑柱是否可以接受这些还原剂，比如说镍柱（His-tag亲和纯化）就会被高浓度的还原剂还原，虑柱颜色由蓝变绿，导致虑柱失活。

稳定剂

一般来说，除了上面提到的四种元素外，我们还可以向蛋白缓冲液中加很多东西，去帮助提高蛋白这种缓冲液中的溶解度和在稳定性，小编就统称这些东西为稳定剂。稳定剂的种类、多少等等都要依情况而定。举几个例子，加入BSA（bovine serum albumin）有时候确实可以稳定住其他蛋白；有时候，通过加入甘油（glycerol）或PEG（polyethylene glycol）增加缓冲液粘稠度，确实可以增加蛋白的稳定性；有时候甚至可以加入一些清洁剂/去污剂（e.g. Triton 或者Tween），也会增加稳定性。

如何配置适合的蛋白缓冲液，需要我们在实验室不断尝试，并且依照情况灵活改变的。有什么关于配置蛋白缓冲液的想法，欢迎留言讨论哦！

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